Group B streptoccocci (GBS) remain the most significant pathogen causing neonatal sepsis, pneumonia, and meningitis. Serotype III GBS are responsible for the majority of GBS infections in the United States. We have confirmed that the type III specific capsule is a major virulence factor and have investigated the role of the capsule in various stages of pathogenesis of GBS infections, using isogeneic rasposen mutants in capsule synthesis. These mutants have also provided the means for identifying and characterizing the genes important for capsule genes. This proposal will extend our investigation into the organization and function of the capsule genes, using contemporary genetic techniques. We have identified the gene cpsF which encodes the enzyme will be used to confirm the transposon mutations previously derived in this gene. Complementation analysis with the wild type cpsF gene will be used to demonstrate its activity in trans. In Aim II, we will identify and characterize open reading frame adjacent to cpsF which may be involved in sialic acid metabolism for capsule synthesis. We will also attempt to clone the sialic acid synthase gene responsible for the endogenesis synthesis of sialic acid by GBS. The last 2 aims will focus on the characterization of gene involved in the synthesis and transport of the oligosaccharide repeating unit and possible role a lipid intermediate in subunit assembly. AimIII will extend our characterization of cpsD, which encodes a galactosyl transferase. These studies will also include the characterization of lipid intermediate during oligosaccharide synthesis. In Aim IV, we will identify and characterize open reading frames, adjacent to cpsD, for their role in oligosaccharide assembly or transport. This research will involve the close collaboration of Dr. Michael Wessels and coworkers through the IRPG, by providing capsule gene mutants, mutant constructs containing cloned recombinant capsule genes for complementation studies, wild type recombinant capsule genes in expression vectors, and mutated capsule genes for complementation for characterizing the biochemical pathways of capsule biosynthesis. The biochemical analysis of the gene products will accelerate the progress in my laboratory on the genetics of capsule expression. Knowledge of the genetic and biochemical basis for the synthesis, transport, and secretion of the capsular polysaccharide will provide a foundation for investigating the regulation of capsule expression and will ultimately enhance our understanding of its role during invasive neonatal GBS infections.